RESUMEN
Aldo-keto reductase has been purified 13,000-fold from the lens of the camel (Camelus dromedarius) to a specific activity of 85 U/mg protein. The enzyme is a monomeric protein, exhibiting a Mr = 40,000 upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Camel lens aldo-keto reductase shows a broad substrate specificity, which is strictly dependent on NADPH, and is insensitive to inhibition by Sorbinil and valproate. Aldoses with a carbon chain with more than four residues, as well as glucuronate, are not reduced by the enzyme. On the basis of substrate specificity and sensitivity to inhibition, camel lens aldo-keto reductase appears to be distinct from the so far described aldose, aldehyde and carbonyl reductases.
Asunto(s)
Oxidorreductasas de Alcohol/aislamiento & purificación , Cristalino/enzimología , Oxidorreductasas de Alcohol/metabolismo , Aldehído Reductasa , Aldo-Ceto Reductasas , Animales , Camelus , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Cinética , Peso Molecular , Especificidad por Sustrato , TermodinámicaRESUMEN
Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was purified 38,750-fold to apparent electrophoretic homogeneity from bovine ocular lens. The enzyme appears to be a homotrimer with a molecular weight of 97,000, and displays non-linear kinetics with concave downward curvature in double-reciprocal plots with orthophosphate as variable substrate. The analysis of the kinetic parameters of bovine lens purine nucleoside phosphorylase, determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases, indicates the occurrence of a rapid equilibrium random Bi-Bi mechanism with formation of abortive complexes. The effect of pH on the enzyme activity and on the sensitivity of the enzyme to photoinactivation, as well as the effect of thiol reagents on the enzyme activity and stability, strongly suggest the involvement of histidine and cysteine residues in the active site. From the measurements of the kinetic parameters at different temperatures, heats of formation of the enzyme-substrate complex for guanosine, guanine, orthophosphate and ribose 1-phosphate were determined. Activation energies of 15,250 and 14,650 cal/mol were obtained for phosphorolysis and synthesis of guanosine, respectively.
Asunto(s)
Cristalino/enzimología , Purina-Nucleósido Fosforilasa/aislamiento & purificación , Animales , Bovinos , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Peso Molecular , Fotoquímica , Purina-Nucleósido Fosforilasa/química , Especificidad por SustratoRESUMEN
The ocular lens is an organ which depends mainly on anaerobic processes to obtain the metabolic energy required for the maintenance of its physiological functions. In these circumstances, the purine salvage pathway enzymes, by using preformed purine rings, and allowing the utilization of the activated ribose moiety of nucleosides, might be of relevance as an energy saving device. In this paper we show that the calf lens possesses many enzymes of the purine salvage pathway, with a particularly high specific activity of purine nucleoside phosphorylase (EC 2.4.2.1), and that the isolated lens epithelium can actively convert adenine and adenosine into adenine nucleotides. In addition, as in bacteria and red blood cells, inosine and adenosine in the lens, acting as ribose donors, exert a profound effect on the process of adenine conversion into ATP.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Cápsula del Cristalino/metabolismo , Adenina/metabolismo , Adenosina/metabolismo , Animales , Bovinos , Metabolismo Energético , Epitelio/metabolismo , Inosina/metabolismo , Masculino , Oxidorreductasas/metabolismo , Ratas , Ratas Endogámicas , Transferasas/metabolismoRESUMEN
The signaling activity of anandamide (AEA) is terminated by its uptake across the cellular membrane and subsequent intracellular hydrolysis by the fatty acid amide hydrolase (FAAH). To date, the existence of an AEA membrane transporter (AMT) independent of FAAH activity remains questionable, although it has been recently corroborated by pharmacological and genetic data. We performed confocal microscopy and biochemical analysis in human HaCaT keratinocytes, in order to study the cellular distribution of AMT and FAAH. We found that FAAH is intracellularly localized as a punctate staining partially overlapping with the endoplasmic reticulum. Consistently, subcellular fractionation and reconstitution of vesicles from membranes of different compartments demonstrated that FAAH activity was localized mainly in microsomal fractions, whereas AMT activity was almost exclusively in plasma membranes. These results provide the first morphological and biochemical evidence to support the view that transport and hydrolysis are two spatially and functionally distinct processes in AEA degradation.
Asunto(s)
Ácidos Araquidónicos/análisis , Ácidos Araquidónicos/fisiología , Queratinocitos/citología , Queratinocitos/fisiología , Amidohidrolasas/fisiología , Ácidos Araquidónicos/farmacocinética , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Línea Celular , Membrana Celular/fisiología , Endocannabinoides , Retículo Endoplásmico/fisiología , Humanos , Hidrólisis , Queratinocitos/efectos de los fármacos , Microscopía Confocal/métodos , Alcamidas PoliinsaturadasRESUMEN
In Bacillus cereus purine ribonucleosides and deoxyribonucleosides share a common inducible catabolic pathway, leading to the formation of ribose-5-P or deoxyribose-5-P respectively inside the cell, while the purine ring remains in the external medium. Both ribo- and deoxyribonucleosides are inducers of adenosine deaminase, inosine-guanosine phosphorylase and phosphopentomutase, the enzymes of the catabolic pathway. We now show that deoxyribonucleosides, but not ribonucleosides, induce the aldolase specific for deoxyribose-5-P (2-deoxy-D-ribose-5-phosphate acetaldehyde lyase, EC 4.1.2.4), thus allowing the sugar moiety of exogenous deoxyribonucleosides to be utilized as an energy source.
Asunto(s)
Aldehído-Liasas/biosíntesis , Bacillus cereus/enzimología , Desoxirribonucleósidos/farmacología , Bacillus cereus/efectos de los fármacos , Inducción Enzimática , Cinética , Relación Estructura-ActividadRESUMEN
Bovine lens aldose reductase (alditol:NADP+ oxidoreductase, EC 1.1.1.21) undergoes an oxidative modification, greatly stimulated by high ionic strength, upon incubation in the presence of oxygen radical generating systems (Del Corso, A., Camici, M., and Mura, U. (1987) Biochem. Biophys. Res. Commun. 148, 369-375). The enzyme modification is accompanied by a change in stereospecificity toward the two enantiomers of glyceraldehyde. In particular, the Km for L-glyceraldehyde of the native form increased over 150 times after the enzyme modification, with a decrease in the catalytic efficiency of over 200 times. By contrast, for the D-enantiomer the Km increased only 7 times with respect to the native form, with a concomitant decrease in the catalytic efficiency of only approximately 3 times. This dramatic change in stereospecificity may account for the reported apparent cooperative behavior exhibited also by highly purified electrophoretically homogeneous preparations of aldose reductase.
Asunto(s)
Aldehído Reductasa/metabolismo , Cristalino/enzimología , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Aldehído Reductasa/aislamiento & purificación , Animales , Bovinos , Ditiotreitol/farmacología , Radicales Libres , Gliceraldehído/metabolismo , Cinética , Mercaptoetanol/farmacología , Oxidación-Reducción , Estereoisomerismo , Especificidad por SustratoRESUMEN
Two structurally different forms of bovine lens aldose reductase have been identified. Freshly prepared lens extracts contain an unactivated "b form" (ARb) which is sensitive to inhibition by Sorbinil. Upon incubation of the extracts with oxygen radical generating systems, ARb is converted to a more active "a form" (ARa), which is not inhibited by Sorbinil. ARa and ARb were purified to electrophoretic homogeneity.
Asunto(s)
Aldehído Reductasa/aislamiento & purificación , Imidazolidinas , Isoenzimas/aislamiento & purificación , Cristalino/enzimología , Deshidrogenasas del Alcohol de Azúcar/aislamiento & purificación , Aldehído Reductasa/antagonistas & inhibidores , Animales , Bovinos , Cromatografía/métodos , Ácido Edético/farmacología , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Imidazoles/farmacología , Hierro/farmacología , Isoenzimas/antagonistas & inhibidores , Extractos de Tejidos/análisisRESUMEN
Among a series of purine nucleosides, inosine was found to be phosphorylated at the highest rate by crude extracts of the cyanobacterium Spirulina platensis. The inosine phosphorylating activity could be separated from hypoxanthine-guanine phosphoribosyl transferase. This result shows that IMP formation may occur via the direct phosphorylation of inosine at its 5'-position, rather than via inosine phosphorolysis, followed by hypoxanthine phosphoribosylation, and provides unequivocal evidence for the occurrence of inosine kinase in nature.
Asunto(s)
Cianobacterias/enzimología , Inosina Monofosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Electroforesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Purinas/metabolismoRESUMEN
The enzymes of the polyol pathway, namely aldose reductase and sorbitol dehydrogenase, were measured in camel lens extracts. A NADPH-dependent glyceraldehyde and erythrose reductase activity 25 times higher than that of calf lens was observed in camel lens. A preliminary comparison between this enzyme activity present in the camel and aldose reductase of calf lens is reported.
Asunto(s)
Aldehído Reductasa/metabolismo , Gliceraldehído/metabolismo , L-Iditol 2-Deshidrogenasa/metabolismo , Cristalino/enzimología , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Animales , Camelus , Bovinos , NADP/metabolismo , Oxidación-Reducción , Especificidad de la EspecieRESUMEN
Bovine lens aldose reductase (alditol: NADP+ oxidoreductase, EC 1.1.1.21) undergoes a modification induced by 2-mercaptoethanol in the presence of the redox system Fe(II)/Fe(III). The modified form (ARa) exhibits an increased hydrophobicity and tendency to aggregate. Moreover, while the native enzyme form is rather insensitive to proteolytic breakdown, the modified form is susceptible to limited proteolysis by trypsin and chymotrypsin. With both proteases, the degradation correlated with a loss of enzyme activity and results in the appearance of one molecular species of 26 KDa (for chymotrypsin) and two molecular species of 24 and 17 KDa (for trypsin). The decline in solubility and the increase in susceptibility to proteolysis of ARa suggests that the thiol-dependent metal-catalyzed modification is comparable to other oxidative systems that mark proteins for degradation.
Asunto(s)
Aldehído Reductasa/metabolismo , Quimotripsina/metabolismo , Hierro/farmacología , Cristalino/enzimología , Mercaptoetanol/farmacología , Tripsina/farmacología , Aldehído Reductasa/química , Animales , Bovinos , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Cinética , Peso Molecular , NADP/farmacología , Fragmentos de Péptidos/aislamiento & purificación , Conformación ProteicaRESUMEN
Analysis by HPLC of the protein-free supernatant obtained after denaturation of aldose reductase shows that the native form of the enzyme (ARb) contains a tightly bound NADP+, which is absent in the oxidatively modified form (ARa). The absorption, fluorescence, and circular dichroism spectra of ARb and ARa are consistent with the presence of the cofactor only in the native form of aldose reductase. On the other hand, the modified enzyme, in appropriate thiol reducing conditions, can tightly bind NADP+. This indicates a potential reversibility of the modification of aldose reductase, at least in terms of retention of the cofactor.
Asunto(s)
Aldehído Reductasa/metabolismo , Cristalino/enzimología , NADP/metabolismo , Aldehído Reductasa/aislamiento & purificación , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Ditiotreitol/farmacología , NADP/aislamiento & purificación , NADP/farmacología , Unión Proteica , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Bovine lens aldose reductase (alditol: NADP+ oxido-reductase, EC 1.1.1.21) undergoes a thiol-dependent oxidative modification catalyzed by the Fe(II)/Fe(III) redox system. The enzyme is inactivated by various oxygen radical generating systems. However, addition of 2-mercaptoethanol to the oxygen radical generating systems resulted in an initial increase followed by a decrease in the activity of aldose reductase. The net maximal increase in the enzyme activity was observed with 3 mM 2-mercaptoethanol, 0.3 mM FeSO4, and 0.9 mM EDTA, either with or without 1 mM hypoxanthine and 37 mU/ml of xanthine oxidase. The formation of the stable, activated intermediate, ARa, appears to proceed through the reaction between the enzyme and the oxidized form of 2-mercaptoethanol which in the presence of iron, forms a mixed disulfide with a cysteine residue. Reduction of ARa with dithiothreitol released 0.7 mol of 2-mercaptoethanol per mole of enzyme and converted it to a form that resembled the native aldose reductase.